The mechanisms by which the immune system distinguishes normal developmental cell death from pathological immunogenic cell killing are central to effective cancer immunotherapy. The goal of this project is to understand the mechanisms by which tumor cell death can induce immune activation, and exploit these mechanisms to develop novel cytotoxic gene therapies for cancer. Preliminary data indicates that the mode of cell death is critical to generation of effective anti-tumor immune responses, such that cells dying via non-apoptotic mechanisms generate greater immune activation than cells dying via apoptotic mechanisms. We have also shown that macrophages are a key cell distinguishing between tumor cells dying through classical apoptosis and cells engineered to die through non-apoptotic mechanisms, and these differential responses of macrophages can be used to cure mice of established tumors. These data indicate that there are specific characteristics of non-apoptotic cell killing that result in immune activation. We aim to develop novel cytotoxic gene therapies that combine the key features of non-apoptotic cell killing, including release of cell contents, activation of cellular stress programs, and generation of an inflammatory tumor environment. To achieve this aim, in combination with candidate cytotoxic genes we will develop a novel approach in gene therapy that involves delivery of transcription factors and signaling molecules to activate broad- spectrum cellular gene expression programs. In view of preliminary data indicating that expression of cell stress- related genes such as Hsp70 by tumor cells during cell killing is key to immune activation, we aim to activate, by genetic means, programs of cell stress in tumor cells. Additionally in view of data indicating that immune activation is achieved in a pro- inflammatory cytokine environment generated by macrophages exposed to non-apoptotic tumor death, concomitant with cell death we aim to activate the broad immune cascade characteristic of the anti-viral response. We hypothesize that this combination of non-apoptotic cell death in a manner and an environment optimal for immune activation will result in increased efficacy of cytotoxic gene therapy approaches. Thus through increased understanding the mechanisms by which immune processes are activated by cytotoxic modalities we will significantly overcome some of the major current limitations of cancer gene therapy; namely limited efficiency of gene delivery and limited efficacy of in vivo cellular cytotoxicity.